Objectives. Alzheimer's Disease (AD) Drug Discovery. Several lines of evidence indicate that amyloid-related protein processing producing toxic oligopeptides including Abeta42 and Abeta40 is associated with neuronal death in AD. There are several enzyme complexes involved in oligopeptide production including alpha-, beta-, and gamma-secretases. The gamma-secretase is associated with toxic oligopeptide production. In this one-year proposal we will abrogate the expression of Presenilin-1 (PS-1) (an important constituent of the gamma secretase) using ribozymes (Rbzs) specific for the PS-1 mRNA inserted in a recombinant Adeno-Associated Virus (rAAV) vector. We will produce and test PS-1-specific Rbzs that successfully inhibit PS-1 in cell free and neuroblastoma cell cultures. Finally testing will be done in human neurons. We hypothesize that PS-1-specific recombinant hammerhead Rbzs will inhibit PS-1 mRNA production by AAV delivery in human neuron cell cultures. Specific Aims 1. To prepare human neuroblastoma and human primary adult and fetal neuron cell cultures and to determine levels of Presenilin-1 (PS-1) mRNA under basal conditions in these cultures. 2. To design and engineer human PS-1 site-specific hammerhead Rbzs and to evaluate their efficiency to inhibit PS-1 mRNA expression in human neuron cell cultures using the Rbz inserted in AAV. Neuroblastoma cell lines will be used to first optimize the molecular procedures. Our Long-Range Goal is to assess Rbz-AAV delivery methods to the brain. In future proposals we could graft autologous genetically engineered cells into animal model brains and ultimately into AD patients. The Rbz-AAV we produce may enter neurons directly and efficiently when introduced directly into the brain. Thus, our proposal is a step in this direction and specifically pertinent to the known PS-1-related injury in AD.